The tooth is an organ having enamel as its outermost layer, a hard tissue called dentin inside the layer, and furthermore, odontoblasts forming the dentin on the inner side with dental pulp in the center. Teeth may be lost due to tooth decay and periodontal disease, but because the presence of teeth has a big impact on one's appearance and on the taste of foods, the concern about tooth reproduction techniques has been increasing. Furthermore, concern towards about tooth reproduction techniques has also been increasing for reasons such as maintaining health and maintaining a high quality of life.
A tooth is a functional unit that is formed by the inducement of the generation process during the fetal period, and is formed by a plurality of cell species. A tooth is not generated by a stem cell system wherein cell species are generated from stem cells such as hematopoietic stem cells and mesenchymal stem cells in adults, and currently, teeth therefore cannot be regenerated with only transplantation of stem cells (stem cell transplantation) achieved by regenerative medicine. Although regeneration of teeth through identification of genes found specifically in the generation process of teeth and artificial inducement of a tooth germ has been examined, complete inducement of regeneration of teeth cannot be achieved only by identifying the genes.
Thus, in recent years, a method for obtaining regenerated teeth by reconstructing the tooth germ using an isolated tissue and cell derived from the tooth germ, and then transplanting the reconstructed tooth germ has been examined.
The present inventors figured out that by arranging a first cell mass and a second cell mass in contact with each other inside a support carrier made from collagen gel, wherein at least either the first cell mass is formed substantially from only one of either mesenchymal cells or epithelial cells derived from the tooth germ or the second cell mass is formed substantially from only the other type of cells, and then by culturing the first and the second cell mass inside the support carrier, cell differentiation can be induced effectively, and it is possible to produce a regenerated tooth germ and regenerated tooth having a specific cell arrangement and directionality (for example, see Patent Literature 1).
Furthermore, the present inventors showed that a regenerated tooth germ and regenerated tooth having a specific cell arrangement and directionality can similarly be obtained even by using oral epithelial cells and their first stage cultured cells as epithelial cells (for example, see Patent Literature 2), or by using amnion-derived cells as mesenchymal cells (for example, see Patent Literature 3), or else by using cells obtained by differentiation inducement of totipotent stem cells as mesenchymal cells (for example, see Patent Literature 4).
Also, in a regenerated tooth germ and regenerated tooth, the size of the tooth differs depending on its position and this size also varies with individuals. Therefore, it is important to control the size from the point of view of regenerating a tooth suitable to the position of the lost tooth. However, in the above documents, the control method for the size of regenerated teeth has not been examined. Furthermore, a set of regenerated teeth may be also obtained by the above method. In such a case, each tooth is separated from the set and used as a graft, but the difficulty in controlling the number of teeth and also the size of each tooth included in the set of teeth is easily expected from the point of view of insufficient development of the three-dimensional cell operation technology and insufficient understanding of the mechanism of form control in development biology.
The method for inoculating a cell mixture of a tooth germ including mesenchymal cells derived from the tooth pulp that form the dental bulb and dentin, and also including epithelial cells that contribute to the formation of the enamel in a scaffold made by solidification of a biodegradable polymer made from a copolymer of polyglycolic acid and polylactic acid, and then transplanting it into the body of an animal to form a tooth is also proposed as a method for producing regenerated teeth having the desired size and shape. In this method, the control of the shape of the tooth has been tested by using a scaffold of the desired shape. However, the regenerated tooth is derived from a tooth germ made from an epithelial cell layer and a mesenchymal cell layer, and the tooth germ is known to grow due to the chronological epithelical-mesenchymal interaction that occurs between the epithelial cells and mesenchymal cells. Thus, if a scaffold is used, sufficient cellular interaction is not obtained. Therefore, the use of a scaffold may not be preferable (for example, see Non-Patent Literature 1). Furthermore, the speed of formation of the tooth is faster than the time taken for the scaffold to decompose. Therefore, the tooth may be formed with some part of the scaffold mixed therein, and it is expected that the reproducibility of the cell arrangement and tooth shape may not necessarily be high.
On the other hand, it was believed that in general, the normal shape of the tooth crown cannot be obtained unless the mesenchymal tissue in the reconstructed tooth germ is perfect. However, it has been reported that even if a mesenchymal cell mass (mass obtained by centrifugal processing after separating the tissues with enzyme treatment) is used in place of mesenchymal tissue, when the number of cells is larger, a comparatively larger size of tooth germ is obtained in the in vitro culture, and the number of tooth cusps is also increased (for example, see Non Patent Literature 1). However, even after transplanting this tooth germ inside a living organism, the shape of the tooth crown and the number of tooth cusps change as compared to a normal tooth, and a tooth with the correct shape is not obtained. According to the report, a tooth with a correct shape is not obtained even by reconstructing through the combination of an epithelial cell mass and a mesenchymal cell mass. According to the report, in the reconstructing using an epithelial tissue and a mesenchymal cell mass, the correct shape can be created if mesenchymal tissue can be used, however, the limitation imposed during the producing of teeth regarding the use of unified mesenchymal cells indicates that partial resolution is achieved by increasing the number of cells.
Furthermore, it has been reported that when a reconstructed tooth germ is produced with an epithelial tissue and a mesenchymal cell mass, the number of produced teeth increases when the number of mesenchymal cells is increased, however, the size of the teeth is not affected (Non-Patent Literature 2). In the report, the final size of a tooth and tooth cusp is determined by intrinsic factors of the mesenchymal tissue and epithelial tissue, that is, it is concluded that the mesenchymal cells and epithelial cells have an intrinsic memory concerning the final size of a tooth and tooth cusp, respectively.
Furthermore, the present inventors figured out a method for using as many epithelial tissues in the region configuring the enamel knot as the desired number of teeth to form an epithelial cell aggregate, when producing a reconstructed tooth germ using an epithelial cell aggregate and a mesenchymal cell aggregate as a method for controlling the number and form of the teeth to be produced (Patent Literature 5). According to the method, an aggregate of teeth having a desired number of teeth can be obtained. However, as many epithelial tissues as the number of the desired teeth must be acquired to constitute the region configuring the enamel knot. Furthermore, Patent Literature 5 does not examine the control of the size of a tooth.